The broad objective of this proposal is to understand the biochemical events that comprise the process of genetic recombination. This goal will be achieved by reconstituting various steps of the recombination process in vitro, using purified proteins from Escherichia coli and from Saccharomyces cerevisiae, and also by understanding the mechanism of these molecular events using defined biochemical reactions. Genetic recombination is a fundamental biological process that involves the processing of broken DNA, homologous recognition, exchange of DNA strands, and resolution of the recombination intermediates. It is an important cellular process that is used by all organisms to repair DNA damage, restart DNA replication, and generate genetic diversity. Two specific objectives are planned. The first set of aims is to fully reconstitute in vitro the genetic recombination process of E. coli. The second major aim is to determine the biochemical mechanism of steps that define recombination in the eukaryote, S. cerevisiae. Understanding the mechanism of recombination will provide insight into the manner by which DNA breaks are repaired and, thereby, ensure that genomes are accurately repaired and maintained. Failure to repair DNA properly can ultimately lead to cancer development. The human homologs of some of the proteins that we will investigate include the Breast Cancer Susceptibility Gene 2 (BRCA2) and the Bloom (BLM) helicase. Understanding the mechanism of recombination should also provide insight into possible new experimental approaches for gene replacement therapies.